1. Technical Field
The present invention relates to methods for culturing bacteria and other culturable microorganisms to measure the germ counts. More specifically, it relates to a microorganism analyzing method in which the germ count can be measured quickly and in a highly sensitive manner, even in samples that include contaminants other than microorganisms.
2. Description of Related Art
The conventional method that has generally been used to culture microorganisms and to measure their number is the agar plate smear method. In this method, a prescribed amount of a specimen is smeared onto an agar plate culture, which contains appropriate nutrients. The specimen is cultured until the colony is large enough to be observed macroscopically, and then the resulting colony is measured. When this method is used, however, one must wait until the bacterial colony grows to a size that is large enough to be observed macroscopically. Therefore, a culture period of 18 to 24 hours is generally the extent required for inferring what the category of bacteria is. Depending on the type of bacteria, there are some that require a culture period in excess of 24 hours, in excess of 48 hours, and even those that require a long-term period extending one month.
Depending on the type of bacteria that are grown in the culture, there are occasions that require germ-type identification screening to single out microorganisms, or that require drug sensitivity tests to examine the efficacy of certain drugs. With the conventional method, however, judging the need for these tests takes time; moreover, efficiently screening a large number of samples is difficult.
Instead of agar plate culture, samples can be cultured by a method employing liquid culture. According to this method, a fixed volume of the specimen is combined with a liquid culture that contains the appropriate nutrients; culture is thus performed, and the turbidity is measured macroscopically, or by means of an absorption photometer or a spectrophotometer. Photometers are not known to have fine sensitivity; thus, when this method is used, one must wait for the growth of the bacteria, until there is a change in the turbidity. For this reason, the measurement of the number of bacteria, as in the case of the agar smear plate method, requires several days of culture. This results in the aforementioned problems.
Moreover, Japanese Laid-open Patent Application No. 5-82901 discloses a method for counting bacteria by irradiating the specimen in the culture with light and by observing the changes over time of the scattered light and the transmitted light. By using this method, one can find accurate germ counts, even wherein the germs differ in developmental form. Since this method utilizes the changes in the scattered light data and the changes in the transmitted light data as the bacteria grows, the time needed for the specimen culture cannot be shortened.
Meanwhile, particle measurement devices, flow cytometers in particular, are known to be highly sensitive devices for measuring extremely small particles, such as microorganisms. Since particle measurement devices calculate each and every particle in a sample, it is possible to perform highly sensitive measurements. Accordingly, by using a particle measurement device, it is possible to measure the number of bacteria in the sample, without long hours for culturing.
Particle measurement devices, however, detect all particles that are the size of microorganisms, including contaminants such as dust and precipitants, as microorganisms. Thus, detection results of particle measurement devices invariably contain errors caused by the measurement of contaminants. When urine, for example, is used as a specimen and is measured for bacteria, the components in the urine that have such physical forms, as erythrocytes, leukocytes, epitheliocytes, casts, crystals and fragments thereof, are detected as bacteria.
Therein, to differentiate between contaminants and microorganisms, a method of staining microorganisms with fluorescent dyes and measuring the fluorescence they emit has been advocated. The degree of staining, however, differs according to the type of microorganism. Moreover, fluorescent dyes and treatment conditions for staining only the targeted microorganisms must be established according to each sample. Thus, a great deal of trouble is required to perform the analysis; this staining method is not suited to assaying large quantities of specimens.
Other methods for the rapid detection of microorganism counts include: measuring changes in the impedance of the culture that accompanies the growth of the microorganisms, measuring changes in the pH of the culture fluid, and measuring amounts of consumed oxygen or carbon dioxide produced, and research has recently been conducted on methods of finding microorganism counts from correlations between these measurements and microorganism counts. It is possible, however, that these measurement values will change due to causes apart form the microorganisms cultured. Moreover, when considering limitations in the microorganisms that can be detected and the degree of accuracy, these methods cannot be considered satisfactory. These methods can only be used under special conditions. In other words, as has been shown above, there is no method as yet that is capable of conducting the measurement of microorganisms in specimens both accurately and in a brief period of time.